Fig 1: Correlation between BCAT1 and PAK1 expression and their prognostic value. (A) GSEA terms of the genes whose expression are positively correlated with PAK1 and BCAT1 expression in CLL from RNA-seq datasets. Twenty signalling pathways were activated both in PAK1 and BCAT1 related genes and two were suppressed. (B) The 20 pathways that activated in PAK1- and BCAT1-related gene sets. NES, normalized enrichment score. (C) Correlation between PAK1 and BCAT1 levels in CLL patients based on bulk RNA-seq data. Pearson's correlation coefficient values (r) and P values are indicated. Kaplan–Meier analysis of the correlation between PAK1 expression (D), BCAT1 expression (E) or their combined expression (F) and overall survival (OS) in 53 patients with CLL. Log rank tests were used to determine statistical significance. (G) Univariate analysis to identify factors corresponding to the expression of PAK1 and BCAT1. Chi-squared test, Pearson's test and Wilcoxon–Mann–Whitney test were used according to the types of data. *P < 0.05, **P < 0.01, ***P < 0.001. [Colour figure can be viewed at wileyonlinelibrary.com]
Fig 2: Dysregulation of PAK1 and BCAT1 in ibrutinib-resistant cells. (A) Correlation map of chromosome 11 at a resolution of 1 m. Lower panels indicate bins that switched compartments. The indicated regions (red dashes) represent PAK1 loci. (B) Correlation map of chromosome 12 at a resolution of 1 m. Lower panels indicate bins that switched compartments. The indicated regions (red dashes) represent BCAT1 loci. Matrix of normalized differences in correlation coefficients between MEC-1 and MEC-1R cells for chromosome 11 (C) and chromosome 12 (D). Lower panels show the RNA-seq analysis of the two groups. (E) Volcano plot comparing the expression fold changes of proteins between MEC-1 and MEC-1R cells based on TMT data. Multi-omics profiling was performed with three independent biological replicates of MEC-1 and MEC-1R cells. (F–H) RT-qPCR and western blot analysis showed the higher level of PAK1 and BCAT1 in MEC-1R cells compared to MEC-1 cells (N = 3) (mean ± SD). **P < 0.01and ***P < 0.001 by Student's t test. [Colour figure can be viewed at wileyonlinelibrary.com]
Fig 3: Expression and clinical prognostic value of PAK1 and BCAT1 in CLL patients. (A) Expression of PAK1 in CLL patients (n = 15, mean ± SD) compared to normal controls (n = 55, mean ± SD), as determined via RT-qPCR. NC, normal controls (CD19+ B cells from healthy volunteers). P values were calculated using the two-tailed t-test statistical analysis. (B) BCAT1 mRNA levels in CLL patients compared to normal controls. P values were calculated using the two-tailed t-test statistical analysis, error bars indicated SD. (C) Scatter plots showing the correlation between PAK1 and BCAT1 mRNA expression (N = 55). (D) GSEA of PAK1-related genes. (E) The protein levels of PAK1, BCAT1 and mTORC1 signalling molecules in CLL patients and normal controls. TN, treatment naive; R/R, refractory or relapsed. All experiments were performed in triplicate. (F) The quantitative analysis of protein bands of PAK1 and BCAT1 and correlation coefficient was calculated (N = 12). Kaplan–Meier analysis of the correlation between PAK1 expression (G), BCAT1 expression (H) or their combined expression (I) and overall survival (OS) in the validated cohort of 53 CLL patients (N = 50; survival data of three patients was now available), Log rank tests were used to determine statistical significance. *P < 0.05, ***P < 0.001, ****P < 0.0001. [Colour figure can be viewed at wileyonlinelibrary.com]
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